Members of Flavivirus genus are the most important arthropod-borne viruses causing disease in humans. This genus includes viruses (West Nile virus (WNV), Japanese encephalitis virus (JEV) and Dengue virus (DENV)) that are re-emerging and becoming endemic in new areas of the world. Flaviviruses account for ~100 millions infections per year, with billions at risk and no specific therapy available. Although interferon (IFN) responses control the cell and tissue tropism of WNV and other flaviviruses, the specific effector molecules that restrict infection remain poorly characterized. The studies in this collaborative and inter-disciplinary project between the Diamond and Chanda laboratories will use genetic screens to identify novel interferon stimulated genes (ISG) that modulate flavivirus infection in specific cell types ex vivo and in vivo. Loss-of-function high-throughput genetic screens will be performed with a custom- generated GFP-marked shRNA library targeting ~700 mouse and human ISGs, to identify novel ISG that restrict infection of virulent and attenuated strains of WNV. Using stable cell lines that have targeted reductions or ectopic expression of candidate ISGs, we will define mechanistically how novel ISG effector molecules restrict specific steps in the viral lifecycle and influence infection outcome. In preliminary studies, we have identified several candidate inhibitory ISG (Ifi27, IFIT2, and IFITM3) that attenuate WNV infection, and already acquired or generated knockout mice. Using these mice, we will evaluate the function of these particular ISGs in restricting WNV replication in a cell-specific manner and in vivo. We hypothesize that specific ISGs have antiviral properties that differentially control WNV infection and spread, and that these demonstrate tissue and cell-type specificity. Overall, these experiments will more clearly define the interface between IFN control and flavivirus pathogenesis and possibly, guide strategies that modulate immunity to infection by this family of viruses.